Heterocyclic volatiles formed by heating cysteine or hydrogen sulfide with 4-hydroxy-5-methyl-3(2H)-furanone at pH 6.5

The reaction of 4-hydroxy-5-methyl-3(2H)-furanone (HMF) with cysteine or hydrogen sulfide at pH 6.5 for 60 min at 140 degrees C produced complex mixtures of volatile compounds, the majority of these containing either sulfur or nitrogen. Of the 68 compounds detected, 63 were identified, some tentatively, by GC-MS. Among the identified compounds were thiophenes (10), thiophenones (6), thienothiophenes (5), thiazoles (5), trithiolanes (4), pyrazines (6), and oxazoles (4). More compounds were produced in the reaction of HMF with cysteine (63) than were formed in the reaction with hydrogen sulfide (33). In both systems, thiophenones were major reaction products, accounting for 25-36% of the total volatiles formed. Possible reasons for the differences in the composition of the two systems are discussed. The contributions of these reactions, and their products, to the flavor of heated foods are considered.     

Reinvestigation of the reaction between 2-furancarboxaldehyde and 4-hydroxy-5-methyl-3(2H)-furanone

The reaction between 2-furancarboxaldehyde and 4-hydroxy-5-methyl-3(2H)-furanone was reinvestigated as a part of a systematic study on low molecular weight colored compounds from the Maillard reaction. In acetic acid/piperidine, besides 2-(2-furanylmethylene)-4-hydroxy-5-methyl-3(2H)-furanone (1) and 5-[2-(2-furanyl)ethenyl]-2-(2-furanylmethylene)-4-hydroxy-5-methyl -3( 2H)-furanone (2), four novel compounds, 15a, 15b, 16a, and 16b, were isolated and characterized. These compounds are produced from two molecules of furanone 1 and one molecule of 2-furancarboxaldehyde, and a mechanism is proposed for their formation. Compounds 1, 15a, 15b, 16a, and 16b are formed also by reacting 2-furancarboxaldehyde and 4-hydroxy-5-methyl-3(2H)-furanone in water at pH 3 and 2, whereas 2 was never detected. The formation of these compounds was studied also in xylose/lysine and xylose/glycine model systems.     

Formation of 5-methyl-4-hydroxy-3[2H]-furanone in cytosolic extracts obtained from Zygosaccharomyces rouxii

Formation of the flavor compound and precursor 4-hydroxy-5-methyl-3[2H]-furanone (HMF, norfuraneol) was demonstrated in cytosolic protein extracts obtained from Zygosaccharomyces rouxii after incubation with a number of carbohydrate phosphates. 4-Hydroxy-5-methyl-3[2H]-furanone was produced from d-fructose-1,6-diphosphate, d-fructose-6-phosphate, d-glucose-6-phosphate, 6-phosphogluconate, d-ribose-5-phosphate, and d-ribulose-1,5-diphosphate. Enzyme assays revealed d-fructose-1,6-diphosphatase, phosphohexose isomerase, d-glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activity in the cytosolic extracts. Model studies showed the spontaneous formation of HMF from d-ribulose-5-phosphate. It is assumed that d-ribulose-5-phosphate is generated in cytosolic extracts by the action of the investigated enzymes from the carbohydrate phosphates and is then chemically transformed to HMF. The hypothesis was proven by the production of HMF in solutions containing commercially available enzymes and [6-(13)C]-d-glucose-6-phosphate.     

Formation of 2,5-dimethyl-4-hydroxy-3(2H)-furanone through methylglyoxal: a Maillard reaction intermediate

The caramel-like aroma compound, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) was quantified and verified by HPLC and GC-MS in the Maillard reaction based on methylglyoxal (MG). The reaction was performed in the 0.5 M phosphate buffer by heating MG with or without either glycine or cysteine at 120 degrees C for 1 h. MG alone or MG with cysteine could produce increased level of DMHF with pH increased, whereas MG with glycine had contrary trend. Experiments using a 1:1 mixture of [(13)C6]glucose and [(12)C6]glucose indicate that in the presence of glycine or cysteine, glucose skeleton kept intact during DMHF formation since a 1:1 mixture of [(13)C6]DMHF and [(12)C6]DMHF was formed. Acetylformoin was detected in the glucose with amino acid reaction system as a precursor of DMHF, while in the MG reaction systems, acetylformoin could not be identified. It is suggested different pathways of DMHF formation via MG and glucose.     

4-hydroxy-2,5-dimethyl-3(2H)-furanone formation by Zygosaccharomyces rouxii: effect of the medium

The formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) by Zygosaccharomyces rouxii was studied in yeast-peptone-dextrose medium containing d-fructose 1,6-diphosphate under various culture conditions. Cell growth and HDMF production was heavily dependent on medium pH and sodium chloride concentration. Higher pH values of the nutrient medium had a positive effect on HDMF formation but retarded cell growth resulting in an optimal pH value of 5.1 with regard to the yield of HDMF. Salt stress stimulated HDMF formation by Z. rouxii as increasing sodium chloride concentration led to higher amounts of HDMF. The HDMF concentration in the culture supernatant and HDMF formation per yeast cell peaked at 20% sodium chloride in the nutrient medium. The nonutilizable carbohydrate d-xylose displayed a weak effect on HDMF formation, and the addition of glycerol to salt-stressed cells had no effect on the production of HDMF.     

Biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone and derivatives in in vitro grown strawberries

The biosynthesis of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol) and its methyl ether and glucoside derivatives has been studied in strawberries. An in vitro system was used for growing this fruit, showing that the presence in the incubation medium of sucrose or hydroxyquinoline hemisulfate has no effect on the bioformation of these compounds. Strawberries in vitro grown showed an increase in furanone content with time, especially between the second and fourth days, to the same extent as field-grown fruits but at a higher rate. Among the precursors added to the incubation medium, D-fructose gave rise to an increase in furaneol and its glucoside derivative of 42. 6% and 26.3%, respectively. D-fructose 6-phosphate seems to be the precursor of furaneol in strawberries since, when present in the incubation medium, it produced an average increase of 125% in all furanones contents with respect to control fruits.     

Effects of L-cysteine and N-acetyl-L-cysteine on 4-hydroxy-2, 5-dimethyl-3(2H)-furanone (furaneol), 5-(hydroxymethyl)furfural, and 5-methylfurfural formation and browning in buffer solutions containing either rhamnose or glucose and arginine.

Solutions of L-cysteine (Cys) and N-acetyl-L-cysteine (AcCys), containing glucose or rhamnose, with or without arginine, were buffered to pH 3, 5, and 7 and incubated at 70 degrees C for 48 h. Cys and AcCys inhibited the formation of (hydroxymethyl)furfural (HMF) from glucose and methylfurfural (MF) from rhamnose under acidic conditions. AcCys inhibited the accumulation of 4-hydroxy-2, 5-dimethyl- 3(2H)-furanone (DMHF, Furaneol) from rhamnose, but Cys, under our experimental conditions, enhanced Furaneol accumulation from rhamnose. Cys and AcCys reacted directly with Furaneol but not with HMF or MF. Both Cys and AcCys inhibited nonenzymatic browning at pH 7. At pH 3, however, Cys reacted with both glucose and rhamnose to produce unidentified compounds that increased the visible absorbency.     

Identification of 5-hydroxy-3-mercapto-2-pentanone in the maillard reaction of thiamine, cysteine, and xylose

5-Hydroxy-3-mercapto-2-pentanone is claimed in the scientific literature as a key intermediate in the degradation of thiamine and the related generation of aroma compounds; however, there are no analytical NMR and MS data available. We have identified the compound in a thermally treated mixture of thiamine, cysteine, and xylose and characterized it by MS and NMR.     

Influence of human saliva on odorant concentrations. 2. aldehydes,alcohols, 3-alkyl-2-methoxypyrazines,methoxyphenols, and 3-hydroxy-4,5-dimethyl-2(5H)-furanone

The influence of human whole saliva on selected alcohols, aldehydes, 3-alkyl-2-methoxypyrazines, and phenols in food-relevant concentrations was investigated. At pH 7.5-8 it was found that the alcohols, methoxyphenols, methoxypyrazines, and 3-hydroxy-4,5-dimethyl-2(5H)-furanone remained unmodified by saliva, whereas aldehydes were reduced to their corresponding alcohols. Generally, the processes were found to be dependent on the salivary activity of the panelists as well as on the concentration of the applied odorants. Reduction of the aldehydes did not occur after thermal treatment of the saliva. These investigations are aimed at finding an explanation for longer lasting aftertaste in humans, as it is induced by some odor-active compounds after the consumption of food materials.     

Analysis of a model reaction system containing cysteine and (E)-2-methyl-2-butenal, (E)-2-hexenal, or mesityl oxide

Cysteine conjugates, resulting from the addition of cysteine to alpha,beta-unsaturated carbonyl compounds, are important precursors of odorant sulfur compounds in food flavors. The aim of this work was to better understand this chemistry in the light of the unexpected double addition of cysteine to two unsaturated aldehydes. These reactions were studied as a function of pH. When (E)-2-methyl-2-butenal (tiglic aldehyde, 4) was treated with cysteine in water at pH 8, the major product formed was the new compound (4R)-2-(2-[[(2R)-2-amino-2-carboxyethyl]thio]methylpropyl)-1,3-thiazolidine-4-carboxylic acid (6). Under acidic conditions (pH 1), we also observed a double addition, but the second cysteine was linked by a vinylic sulfide bond to form the previously unreported major product, (2R,2'R,E)-S,S'-(2,3-dimethyl-1-propene-1,3-diyl)bis-cysteine (7). When (E)-2-hexenal (12) was treated with cysteine under acidic conditions, the major product was the novel (4R,2' 'R)-2-[2'-(2' '-amino-2' '-carboxyethylthio)pentyl]-1,3-thiazolidine-4-carboxylic acid (13), and the formation of an vinylic sulfide compound analogous to 7 was not observed. Reduction of the acidic crude reaction mixture with NaBH(4) afforded 13 and the cysteine derivative (R)-S-[1-(2-hydroxyethyl)butyl]cysteine (14) in 14% yield. Treating (E)-2-hexenal with cysteine at pH 8 followed by NaBH(4) reduction yielded the new product (3R)-7-propylhexahydro-1,4-thiazepine-3-carboxylic acid (15). Addition of cysteine to mesityl oxide (16), at pH 8, followed by reduction with NaBH(4) furnished (R)-S-(3-hydroxy-1,1-dimethylbutyl)cysteine (3) and the new compound (3R)-hexahydro-5,7,7-trimethyl-1,4-thiazepine-3-carboxylic acid (18).     

Characterization of 2-methyl-4-amino-5-(2-methyl-3-furylthiomethyl)pyrimidine from thermal degradation of thiamin

Thiamin hydrochloride was thermally degraded in phosphate buffer (pH 6.5) at 110 degrees C for 2 h. A major decomposition product was isolated by column chromatography and structurally identified by spectrometric techniques ((1)H NMR, (13)C NMR, 2D NMR, and MS) as 2-methyl-4-amino-5-(2-methyl-3-furylthiomethyl)pyrimidine (MAMP). The possible formation pathway of MAMP was studied using two model systems. It is proposed that MAMP is formed by nucleophilic attack of 2-methyl-3-furanthiol on the thiamin.     

Metabolic fate of isotopes during the biological transformation of carbohydrates to 2,5-dimethyl-4-hydroxy-3(2h)-furanone in strawberry fruits

Isotopically labeled D-glucose, D-fructose, 1-deoxy-D-fructose, and 6-deoxyhexoses were applied to detached ripening strawberry (Fragaria x ananassa) fruits, and the incorporation of the isotopes into the key strawberry aroma compounds 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF, 1) and 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF, 2) was determined by gas chromatography-mass spectrometry. In contrast to previous reports the data clearly showed that 6-deoxy-D-fructose/6-deoxy-D-glucose and 1-deoxy-D-fructose are not natural precursors of the furanones. However, isotopically labeled 1 and 2 were observed after the application of [1-(2)H]-, [2-(2)H]-, and [6,6-(2)H(2)]-D-glucose as well as [U-(13)C(6)]-, [1-(13)C]-, [1-(2)H]-, [6,6-(2)H(2)]-D-fructose. The isotope label of [4-(2)H]-D-glucose was not recovered in the furanones. In contrast, [2-(2)H]-D-glucose was converted to [1- or 6-(2)H]-1 and [1- or 6-(2)H]-2 by the strawberry fruits. The observed isotope shift can be explained by the catalysis of phosphohexose isomerase in the course of the biogenesis of the hydroxyfuranone (1) and the methoxyfuranone (2) from D-glucose. Thus, the applied D-glucose is metabolized to D-fructose-6-phosphate prior to the transformation into the furanones.     

Effect of food reductones, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and hydroxyhydroquinone (HHQ), on lipid peroxidation and type IV and I allergy responses of mouse

The effect of long-term supplementation of food reductones, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) (2%, w/w), detected in many foodstuffs including soy sauce, and hydroxyhydroquinone (1,2,4-benzenetriol) (HHQ) (1.2%, w/w), detected in coffee, on mouse lipid peroxidation and type IV and I allergy responses was investigated. The effect of supplementation of these reductones combined with NO(2) inhalation (5-6 ppm) was also investigated. Levels of thiobarbituric acid-reactive substances in lung were remarkably increased, and those in kidney and liver were slightly decreased by supplementation of DMHF or HHQ. The degree of 2,4-dinitrochlorobenzene (DNCB)-sensitized lymph node cell proliferation as assessed by lymph node assay was remarkably enhanced by supplementation of DMHF or HHQ. Both the DNCB-sensitized and the trimellitic anhydride-sensitized increases in IgE levels of mice were enhanced to greater extent by supplementation of DMHF or HHQ. In no cases were additive effects of NO(2) inhalation observable. Allergen-sensitized type IV and I allergy responses of mice may be enhanced by supplementation of food reductones, DMHF or HHQ.     

Characterization of volatile compounds from the reaction of 3-hydroxy-2-butanone and ammonium sulfide model system

The reactions between 3-hydroxy-2-butanone and ammoniun sulfide at 25, 50, 75, 100, 125, and 150 degrees C were studied. Four well-known flavor compounds, 2,4,5-trimethyloxazole, 2,4, 5-trimethyl-3-oxazoline, 2,4,5-trimethylthiazole, and 2,4, 5-trimethyl-3-thiazoline, were identified. Another four interesting intermediate compounds, 2-(1-hydroxyethyl)-2,4, 5-trimethyl-3-oxazoline, 2-(1-mercaptoethyl)-2,4, 5-trimethyl-3-oxazoline, 2-(1-hydroxyethyl)-2,4, 5-trimethyl-3-thiazoline, and 2-(1-mercaptoethyl)-2,4, 5-trimethyl-3-thiazoline, were also identified by GC-EIMS and GC-CIMS. All these intermediate compounds were formed at 25 degrees C. On the other hand, tetramethylpyrazine was the major product with a reaction temperature higher than 100 degrees C.     

3-Hydroxy-4,5-dimethyl-2(5H)-furanone levels in fortified Madeira wines: relationship to sugar content

The maturation of Madeira wines usually involves exposure to relatively high temperatures and humidity levels >70%, which affect the aroma and flavor composition and lead to the formation of the typical and characteristic bouquet of these wines. To estimate the levels of sotolon [3-hydroxy-4,5-dimethyl-2(5H)-furanone] and their behavior over time, 86 aged Madeira wines samples (1-25 years old), with different sugar concentrations, respectively, 90 g L(-)(1) for Boal, 110 g L(-)(1) for Malvazia, 25 g L(-)(1) for Sercial, and 65 g L(-)(1) for Verdelho varieties, were analyzed. Isolation was performed by liquid-liquid extraction with dichloromethane followed by chromatographic analysis by GC-MS. The reproducibility of the method was found to be 4.9%. The detection and quantification limits were 1.2 and 2.0 microg L(-)(1), respectively. The levels of sotolon found ranged from not detected to 2000 microg L(-)(1) for wines between 1 and 25 years old. It was observed that during aging, the concentration of sotolon increased with time in a linear fashion (r = 0.917). The highest concentration of sotolon was found in wines with the highest residual sugar contents, considering the same time of storage. The results show that there is a strong correlation between sotolon and sugar derivatives: furfural, 5-methylfurfural, 5-hydroxymethylfurfural, and 5-ethoxymethylfurfural. These compounds are also well correlated with wine aging. These findings indicate that the kinetics of sotolon formation is closely related with residual sugar contents, suggesting that this molecule may come from a component like sugar.     

Acid hydrolysis of 1,6-dihydro-4-amino-3-methyl-6-phenyl-1,2, 4-triazin-5(4H)-one (1,6-dihydrometamitron).

Metamitron (1) does not undergo hydrolysis at pH 1-8 and up to 5 M H(2)SO(4). The product of its two-electron reduction, 1, 6-dihydrometamitron (2), on the other hand, undergoes at pH <3 relatively fast hydrolysis. The dependence of the measured rate constant on acidity indicates that the completely protonated form (AH(2)(2+)) predominating in strongly acidic media undergoes hydrolysis slower than the species bearing one less proton (AH(+)). The latter most reactive species is present in highest concentration in solutions of pH between 0 and 2. This species is protonated on the 2,3-azomethine bond and yields as final products 2-hydrazino-2-phenylacetic acid (4) and acethydrazide (5). Kinetic, polarographic, and spectrophotometric measurements indicated for the first dissociation an average value pK(a) = -0.8, for the second pK(a) = 0.95. These observations together with the easy reduction of the 1,6-bond in metamitron (1) indicate that in nature the cleavage of metamitron may be preceded by its reduction to 1, 6-dihydrometamitron (2), which is then hydrolyzed. Thus, anaerobic, reductive conditions are likely preferable for the total microbial degradation of metamitron.     

Spectrophotometric Determination of Nickel(II) in Soil and Standard Alloy Samples Using 5-Methyl-2-acetylfuran-4-methyl-3-thiosemicarbazone (5-MAFMT)

A simple and selective spectrophotometric method was developed for the determination of nickel(II) using 5-methyl-2-acetylfuran-4-methyl-3-thiosemicarbazone (5-MAFMT) as a chromogenic reagent forming a yellow-colored complex at pH 9.5. The complex was instantaneous and stable for 5 h. The system obeyed Beer’s law in the concentration range of 0.06–0.60 µg/mL of nickel(II), with a correlation coefficient of 0.999. The molar absorptivity and Sandell’s sensitivity of the complex species were found to be 1.87 × 10⁴ L/mol.cm and 1.2 × 10^–3 µg/cm² at 361 nm, respectively. The limit of detection was 0.0713 µg/mL with the relative standard deviation (RSD) ≤1.0%. The proposed method is rapid, simple, sensitive, and successfully applied for the determination of nickel(II) when present alone or in the presence of other ions that are usually associated with nickel(II). The method was tested for nickel (II) determination in soil and various standard alloy samples. The recovery of nickel(II) in alloy samples using the developed method was >98% confirming the suitability of the method. Comparisons of the results with those obtained using an atomic absorption spectrophotometer for nickel(II) determination also tested the validity of the method at the 0.05 level.     

Degradation studies on benzoxazinoids. Soil degradation dynamics of (2R)-2-O-beta-D-glucopyranosyl-4-hydroxy-(2H)- 1,4-benzoxazin-3(4H)-one (DIBOA-Glc) and its degradation products, phytotoxic allelochemicals from Gramineae

Wheat (Triticum aestivum L.) has been found to possess allelopathic potential and studies have been conduced to apply wheat allelopathy for biological weed control. 2,4-Dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA) is a common product found in wheat, corn, and rye exudates and it can be released to the environment by that way. In this report, the stability of DIBOA is studied in two soils from crop lands of wheat cv. Astron and cv. Ritmo. These varieties were selected by their concentrations of DIBOA and 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) from aerial parts and by the bioactivities of their aqueous extracts in the growth of wheat coleoptile sections. The degradation rate of DIBOA in these soils was measured in laboratory tests during 90 h by high-pressure liquid chromatography methods. These analyses demonstrate that DIBOA was transformed primarily into 2-benzoxazolinone (BOA). This transformation was similar in both soil types with an average half-life of 43 h. The degradation studies for BOA show its biotransformation to 2-aminophenoxazin-3-one (APO) with a half-life of 2.5 days. Therefore, BOA is an intermediate product in the biotransformation from DIBOA to APO in these wheat crop soils and is consistent with previous findings. APO was not degraded after three months in soil, suggesting that its degradation rate in soil is very slow.     

3-Hydroxy-4,5-dimethyl-2(5H)-furanone (Sotolon) causing an off-flavor: elucidation of its formation pathways during storage of citrus soft drinks.

Gas chromatography/olfactometry (GCO) and gas chromatography-mass spectrometry (HRGC-MS) revealed 3-hydroxy-4, 5-dimethyl-2(5H)-furanone (sotolon) to be responsible for the "burnt" and "spicy" off-flavor observed in citrus soft drinks during storage. Among the ingredients of citrus soft drinks, ethanol and ascorbic acid were identified as the essential precursors of sotolon. Two formation pathways were postulated by studies using (2)H (D)- and (13)C-labeled ethanol and ascorbic acid; i.e., sotolon is formed from two molecules of ethanol and carbons 2 and 3 of ascorbic acid (pathway 1), or it is generated from one molecule of ethanol and carbons 3-6 of ascorbic acid (pathway 2).